Monoclonal antibodies (mAbs) capable of neutralizing the infectivity of a broad range of HIV-1 isolates have enabled the discovery of relatively conserved sites on the viral envelope spike that constitute potential vaccine design targets. These mAbs also represent tools to guide said vaccine design. MAb B4e8 binds central to the tip of the V3 loop on the gp120 subunit of the envelope spike and neutralizes a modest range of viruses. The B4e8 epitope occurs somewhat infrequently (~35%) in HIV-1 subtype B, explaining its somewhat modest neutralizing activity. However, variations of the B4e8 epitope, all of which involve a single Arg Gln alteration, occur at substantially higher frequency (>70%) in HIV-1 subtypes other than subtype B. This alteration reduces B4e8 binding affinity considerably and, accordingly, non-subtype B viruses with the Gln are neutralized poorly by B4e8. In contrast, non-subtype B viruses with an Arg are neutralized well. Based on the observations outlined above we hypothesize that the B4e8 segment, i.e. the V3 tip segment equivalent to the B4e8 epitope, may also be accessible on viruses that are recognized poorly by B4e8. The purpose of this grant application is to determine the potential importance of the B4e8 segment on V3 as a vaccine target. In the R21 exploratory phase of this proposal we will pursue 3 aims to address this point. In Aim 1, combinatorial libraries of B4e8 mutants will be constructed and yeast surface display technology will be utilized to identify residues within the paratope that are required for the functional interaction with antigen. In Aim 2, B4e8 variants with higher affinity for non-subtype B V3 sequences will be generated by in vitro antibody evolution and evaluated for neutralizing activity against non-B viruses. In Aim 3, computational protein design will be applied to explore the feasibility of engineering antigens that present the segment of V3 recognized by mAb B4e8 favorably and evaluate the ability of select antigen combinations to promote the elicitation in animals of antibody responses targeted to the B4e8 epitope. Achievement of the objectives in the R21 phase will provide critical insight into accessibility of the B4e8 segment on different HIV subtypes and, thus, have implications for vaccine target discovery. The R33 developmental phase of this proposal focuses therefore on immunogen design and optimization. In Aim 1 of this phase, antigen design improvements will be made, thereby applying insight gained from antibody responses to R21-phase immunogens, and B4e8 variants from the R21 phase will be utilized as templates to design further antigens to target high-frequency non-subtype B V3 sequences not recognized by the parental antibody. In Aim 2, animals will be immunized with these antigens and serum antibody responses will be analyzed for improved cross-neutralizing activities and specificities matching the antibody templates. PUBLIC HEALTH RELEVANCE: An effective vaccine is required to stop the global spread of HIV-1. The first phase of this application seeks to determine whether a relatively conserved segment of a protein on the surface of the virus is accessible to antibodies and whether such antibodies can neutralize the infectivity of a significant range of viral isolates. If so, the identified segment would constitute a promising target for HIV vaccine research and, therefore, in the second phase of application, optimization of the design and testing of experimental vaccine candidates to elicit neutralizing antibodies to the target segment will be pursued vigorously.